Idiopathic thrombocytopenic purpura (ITP) is a common immunohematologic disorder caused by platelet-reactive autoantibodies as described in Bussel et al. (2000, In Hematology: Basic Principle and Practice, pp. 2096-2114, Churchill Livingstone, Philadelphia, Pa.). Briefly, in ITP, the clearance of antibody-coated platelets by tissue macrophages is accelerated, and in some cases, the antibodies also impair platelet production. Childhood-type ITP is self-limiting in about 80% of cases and may be associated with a previous viral infection. Adult-onset ITP is a chronic illness in more than 70% of cases and may occur in association with other disorders, including systemic lupus erythematosus (SLE), lymphoproliferative diseases, common variable immunodeficiency (CVID) disease, and human immunodeficiency virus (HIV) infection.
The decision to treat patients with ITP takes into account the patient's age and disease severity and the anticipated natural history of the disorder. Therapy is initially directed toward impeding the clearance of antibody-coated platelets by using glucocorticoids, splenectomy, anti-blood group D [(anti-Rh(D)] immunoglobulin (Ig), intravenous γ-globulin (IVIG), and other treatments. Immunosuppressive therapy is nonspecific, often toxic, and typically reserved for patients with refractory disease.
Numerous studies have been performed to characterize the pathogenic autoantibodies responsible for platelet destruction and thereby provide a reliable way to diagnose ITP, understand its pathogenesis, and predict responsiveness to therapy. IgG antibodies that react with platelet glycoprotein (GP) IIb/IIIa and GPIb/IX have been identified in some patient serum samples and platelet eluates. See, e.g., van Leeuwen et al. (1982, Blood, 59:23-62), McMillian et al. (1987, Blood 70:1040-1045), Kiefel et al. (1991, Brit. J. Haematol. 79:256-262), and He et al. (1994, Blood 83:1024-1032). However, other platelet antigens also appear to be targeted as described in, e.g., He et al. (1994, Blood 83:1024-1032), Bierling et al. (1994, Brit. J. Haematol. 87:631-633), Hou et al. (1995, Eur. J. Haematol. 55:307-314), Pfueller et al. (1990, Brit. J. Haematol. 74:336-341), Sugiyama et al. (1987, Blood 69:1712-1720), Tomiyama et al. (1992, Blood 79:161-168), Deckmyn et al. (1994, Blood 84:1968-1974), Honda et al. (1990, Brit. J. Haematol. 75:245-249) and Varon et al. (1990, Clin. Immunol. Immunopathol. 54:454-468). In many cases, the antibody specificity cannot be determined or even detected as described in, e.g., Bussel et al. (2000, In Hematology: Basic Principle and Practice, pp. 2096-2114, Churchill Livingstone, Philadelphia, Pa.).
Furthermore, there is no formal proof that any single subset of antibodies, such as, for example, those directed at GPIIb/IIIa, are responsible for platelet destruction. Consequently, previously, the clinical utility of measuring serum or platelet-elutable Ig is unknown and does not have a definitive role in the diagnosis or treatment of ITP or in distinguishing between the adult-onset and childhood-onset forms of the disease as in George et al. (1996, Blood 88:3-40). As a result, the diagnosis of ITP remains one of exclusion and the usefulness of available platelet-antibody tests to confirm or exclude the diagnosis independent of other criteria has not been established (see, e.g., Bussel et al. (2000, In Hematology: Basic Principle and Practice, pp. 2096-2114, Churchill Livingstone, Philadelphia, Pa.).
These prior art limitations illustrate the difficulty involved in characterizing a pathologic autoimmune response by analyzing polyclonal serum. To understand clonality, genetic origin, somatic mutation, and the molecular basis of pathogenicity, repertoires of IgG anti-platelet autoantibodies, e.g., those produced in vitro from the B cells of affected patients, must be studied. Conventional B-cell immortalization approaches for cloning human monoclonal antibodies result in low transformation frequencies and have a propensity for generating IgM-producing clones, thus causing a sampling bias as in Winter et al. (1991, Nature 349:293-299) and Burton et al. (1994, Adv. Immunol. 57:191-280). Consequently, all but one, as in Olee et al. (1997, Brit. J. Haematol. 96:836-845) of the reported human anti-platelet autoantibodies isolated from patients with ITP have been of the IgM class and no more than 2 or 3 unique antibodies have been isolated from a given patient as in Deckmyn et al. (1994, Blood 84:1968-1974), Honda et al. (1990, Brit. J. Haematol. 75:245-249), Nugent et al. (1987, Blood 70:16-22), Hiraiwa et al. (1990, Autoimmunity 8:107-113); and Kunicki et al. (1991, Autoimmunity 4:433-446). Since ITP is an autoimmune disease mediated by platelet autoantibodies of the IgG class, which autoantibodies possess Fc domains and which, unlike antibodies of the IgM class, can interact with receptors on splenic macrophages leading to platelet consumption, the disease relevance of the IgM monoclonals isolated using conventional cell cloning techniques is unclear. Furthermore, the single reported IgG platelet autoantibody derived using cell cloning technique (Olee, above) was found to bind to keyhole limpet hemocyanin as well as to platelet GPIIb/IIIa and demonstrated a three-fold better specificity for tetanus toxoid, thus calling into question the actual specificity of that one purportedly “auto” antibody. As a result, it has been difficult to assess the genetic diversity and other biochemical and immunological properties among ITP-associated autoantibodies within an individual patient, among patients, and in different clinical settings using conventional approaches.
In sum, there are no effective methods of diagnosis or specific treatment modalities for ITP, a disease which causes significant human morbidity and mortality. Despite these long-felt needs, prior obstacles to identifying which, if any, antibodies are potential diagnostic and/or therapeutic targets relating to this disease have prevented development of useful diagnostics and therapeutics for ITP. The present invention meets these needs.
Additionally, platelet aggregation is an essential event in the formation of blood clots. Under normal circumstances, blood clots serve to prevent the escape of blood cells from the vascular system. However, during certain disease states, clots can restrict or totally occlude blood flow resulting in cellular necrosis. For example, platelet aggregation and subsequent thrombosis at the site of an atherosclerotic plaque is an important causative factor in the genesis of conditions such as angina, acute myocardial infarction, and reocclusion following successful thrombolysis and angioplasty.
Heart attack patients are typically treated with thrombolytic agents such as tissue plasminogen activator or streptokinase, which dissolve the fibrin component of clots. A major complication associated with fibrinolysis is reocclusion based on platelet aggregation which can result in further heart damage. Since GPIIb/IIIa receptors are known to be responsible for platelet aggregation, reagents which block the activity of these receptors are expected to reduce or prevent reocclusion following thrombolytic therapy and to accelerate the rate of thrombolysis. Such reagents are also expected to be useful in therapy of other vaso-occlusive and thromboembolic disorders.
One prior art approach to blocking platelet aggregation involves monoclonal antibodies specific for GPIIb/IIIa receptors. A murine monoclonal antibody, designated 7E3, that inhibits platelet aggregation and appears useful in the treatment of human thrombotic diseases is described in published European Patent Application Nos. 205,207 and 206,532, as well as U.S. Pat. No. 5,976,532, to Coller et al. However, it is well-known in the art that murine antibodies have characteristics which severely limit their use in human therapy due to their immunogenicity when administered to a human. Additionally, the need for readministration of such therapeutic modalities in thromboembolic disorders increases the likelihood of these types of immune reactions.
In order to overcome the limitations of administering a mouse antibody to humans, chimeric antibodies consisting of non-human binding regions joined to human constant regions have been produced (e.g., 1984, Proc. Natl. Acad. Sci. USA 81:6851; and PCT Application No. PCT/GB85 00392). However, the technical difficulties associated with such chimeric antibodies (e.g., loss of binding specificity and or avidity, as well as continued immunogenicity when administered to humans) have severely limited their therapeutic applicability in human patients.
Thus, the prior art limitation in production of human anti-platelet autoantibodies, combined with the obstacles in producing murine/human chimeric antibodies to platelet antigens, have prevented the production of human anti-platelet autoantibodies to treat disorders and diseases relating to platelet function, including clotting, despite the long-felt acute need for such therapies. The present invention meets these needs.